Multivalent VLP conjugates

ABSTRACT

The invention is directed to vaccines comprising capsular polysaccharides conjugated to one or more components of virus like particles (VLP), and methods for the administration of and methods for the manufacture of vaccines of the invention. Preferably vaccines of the invention generate a therapeutically effective response in an individual in need thereof to multiple strains and/or serotypes of the same or of different infectious agents. Preferably such vaccines generate a therapeutically effective immune response to all pathogenic strains and/or serotypes of the same infectious agent. In particular, the invention is directed to methods and compositions for the cost efficient administration of a vaccine to a patient in need thereof exposing the patient&#39;s immune system to only the immunogenic components that are likely to be beneficial for the generation of a protective immunological response, both efficacy and safety are increased and cost effectively.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/262,537 filed Sep. 12, 2016, which issued as U.S. Pat. No. 10,159,728on Dec. 25, 2018, which claims priority to U.S. Provisional ApplicationNo. 62/216,646 entitled “Multivalent VLP Conjugates” filed Sep. 10,2015, the entirety of each are hereby incorporated by reference.

BACKGROUND 1. Field of the Invention

The present invention provides compositions, methods and vaccines thatare multivalent for use against viral, bacterial and others diseases, inparticular, compositions and methods for the manufacture of multivalentconjugate vaccines using VLP's.

2. Description of the Background

Infections caused by viral, bacterial or other agents occur in a varietyof animals. Infections are generally species specific and classifiedinto distinct groups based on the host that they infect which caninclude multiple serotypes. Multivalent vaccines have been developed inan attempt to vaccinate against all or the most likely serotypes of theinfectious agent. A multivalent pneumococcal polysaccharide vaccine,Prevnar 13, has been used in preventing pneumococcal disease. Prevnar 13contains thirteen pneumococcal serotypes, serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, and 23F. There still exist nine additionalserotypes that could be pathogenic, and thus, the Prevnar 13 vaccinedoes not address infections caused by these additional nine serotypes.Another problem exists in that these new serotypes do not have a humanhost. As vaccine development takes many years, there exists an urgencyto develop a suitable vaccine as soon as possible.

Virus-like particles (VLPs) have been shown to be useful as vaccinesagainst a variety of infectious agents including viral and bacterialinfections. VLP's are formed from the self-assembly of structuralproteins of selected groups of viruses. These proteins self-assemblyinto a capsule, but, as none of the replicating nucleic acids arepresent, the VLP cannot replicate virus genome and create more orotherwise infectious virus particles. VLPs are strictly non-infectiousand generally harmless to the environment.

When VLPs are formed in the presence of an antigenic molecule, the VLP'sbecome delivery vehicles for the antigen or, in other words, effectivevaccines. VLPs can possess an antigenicity similar to the parent virusfrom which the structural components were obtained or derived andtherefore useful as vaccines against that particular virus infection.VLP's are generally useful as vaccines by possessing antigen within thecomponents of the VLP. This allows for foreign antigens to be exposed ontheir surfaces. Other VLPs have been used as carriers for foreignantigens, including non-protein antigens, via chemical conjugation.However, decorating VLPs with target-antigens by genetic fusion orchemical modification is time-consuming and often leads to capsidmisassembly or antigen misfolding, hindering generation of protectiveimmunity.

Presently available vaccines, such as vaccines against Streptococcuspneumoniae, involve a multivalent polysaccharide conjugate vaccine usinga protein carrier. Currently there is a limitation on the number ofantigen conjugates which creates a zone of no protection to the balanceserotypes. As the current vaccine prevents infection caused by pathogensspecific to the vaccine antigens, pathogens not antigenicallyrepresented in the vaccine become more predominant. In such acircumstance there would be no protective vaccine available for aconsiderable time which would include the time required to identify thenew pathogen, to identify and characterize target antigens, and todevelop a new vaccine. Also the current issue is the single protein usedas a carrier protein. The protein component in the conjugate is twicethe polysaccharide quantity and starts becoming a huge number with anincrease in valency. For multivalent vaccine containing many serotypes,the protein load for the patient can be enormous. For example, assumingthat the polysaccharide antigenic portion would be required at twomicrograms per dose per serotype and four micrograms of carrier proteinper dose per serotype, a 23 valent vaccine would create a protein loadof about 92 micrograms in a dose that could be disastrous for thepatient.

Accordingly, a need exists for a vaccine that does not induce a proteinoverload, but is protective against all pathogenic strains and/orserotypes of a particular infectious microorganism.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantages,associated with current strategies and designs and provides novelvaccines and methods for fighting infectious diseases and, inparticular, all pathogenic strains of a particular disease.

One embodiment of the invention is directed to vaccines comprising oneor more, and preferably multiple immunogenic antigens obtained orderived from the same or a different infectious agent coupled with viruslike particles (VLP). Preferably the immunogenic antigens arepolysaccharides (PS) representing multiple or all pathogenic serotypesof the infectious agent. Preferably the virus like particles areobtained or derived from hepatitis virus, human papilloma virus,respiratory syncytial virus, flavivirus or combinations thereof.Preferably the multiple polysaccharides are covalently coupled viaconjugation to one or more components of the virus like particles.Preferably the conjugation involves coupling via a cyanylating agentsuch as, for example, 1-cyano-4-(dimethylamino)-pyridiniumtetrafluoroborate (CDAP), 1-cyanobenzotriazole (1-CBT),2-cyanopyridazine-3(2H)-One (2-CPO), 1-cyanoimidazole (1-CI),1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) or acombination thereof.

Preferably the infectious agent comprises a virus, a bacterium, or aparasite. Preferably, the virus comprises one or more of enterovirus,hepatitis virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), influenza virus, pertussis virus, rubella virus, tetanusvirus, varicella zoster virus (VZV), flavivirus, West Nile virus, denguevirus, tick-borne encephalitis virus, yellow fever virus, Zika virus andcombinations thereof. Preferably the bacterium comprises one or more ofChlamydia, Clostridium, diphtheria, meningococcal, streptococcal,staphylococcal, pneumococcal, Bacillus or combinations thereof.Preferably the parasite comprises giardia, plasmodium or a combinationthereof. Preferably the VLP are obtained or derived from a single virus,such as for example, a hepatitis virus which may be recombinantlyproduced, but may be produced from multiple different viruses.Preferably the polysaccharide is covalently coupled to one or morecomponents of the virus like particles, and obtained and/or derived fromone or more, two or more or all of the serotypes 1, 2, 3, 4, 5, 6B, 7F,8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and33F of Streptococcus pneumoniae. Vaccines of the invention may comprisean adjuvant such as, for example, aluminum hydroxide with phosphatebuffer, or may not require an adjuvant to generate a therapeuticallyeffective immune response against the infectious agent uponadministration to a patient.

Preferably the vaccine is therapeutically effective against multipleserotypes (e.g., multivalent polysaccharide-VLP conjugate vaccine) andgenerates the humoral and/or a cellular immune response against one ormore infectious agents. Preferably the therapeutically effective vaccineis a vaccine the provided protection to an individual against asubsequent infection and/or provides therapeutic treatment to aninfected individual. Preferably the therapeutically effective vaccinesof the invention provide protection against multiple serotypes of theinfectious agent. Preferably the therapeutically effective immuneresponse comprises a humoral and/or a cellular immune response againstthe infectious agent.

Another embodiment of the invention is directed to methods of preventingor treating infection by the infectious agent comprising administeringthe vaccine of the invention to a patient. Preferably administrationcomprises intramuscular injection, intraperitoneal injection,intravenous injection, intranasal, oral or transdermal. Preferably thepatient is an infant, a toddler, an adolescent, an adult or a senior.

Another embodiment of the invention is directed to methods ofmanufacturing the vaccine of the invention comprising: mixing componentsof virus like particles with multiple immunogenic antigens of the sameor different infectious agents; conjugating the components of the viruslike particle with multiple immunogenic antigens of the same ordifferent infectious agents; and forming a vaccine of conjugated viruslike particles. Preferably the conjugated VLP are lyophilized. Vaccinesof the invention may or may not contain adjuvant. Preferred adjuvants,when used include, for example, aluminum hydroxide and a phosphatebuffer. Vaccines of the invention may further contain a stabilizingagent. Preferred stabilizing agents comprise, for example, sorbitoland/or degraded gelatin.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE INVENTION

Conventional procedures for the development of multivalent vaccinesinvolve multivalent polysaccharides in a protein carrier. Because of thelarge number of strains and/or serotypes of a particular infectiousagent, the protein load can be enormous and, therefore, making them suchconstructions useable as vaccines. When valency (i.e. the number ofstrains, serotypes of the infectious agent) is reduced to addressprotein/antigen overloads, zones of no protection are created topathogenic serotypes unrepresented in the vaccine. As a consequence,pathogens not antigenically represented in the vaccine become morepredominant in the otherwise vaccinated population. Although short termprotection of some of the population may be achieved, the longer termprospect can be the rapid and aggressive generation of infectionsattributed to unvaccinated strains with the expected pathologies andcomplications that now may be otherwise untreatable.

It was surprisingly discovered that virus-like particles (VLP)conjugated with multiple different polysaccharides (PS) representing theserotype variations of a particular infectious agent can be utilized aseffective multivalent vaccines. Surprisingly, even with a large numberof conjugated PS there is little to no capsid misassembly or antigenmisfolding that would otherwise hinder generation of protective immunityfrom the resulting vaccination. Although adjuvant may be added, it wasalso surprisingly discovered that added adjuvant is not necessary for atherapeutically effective and protective vaccine. A therapeuticallyeffective vaccine is a vaccine the provided protection to an individualagainst a subsequent infection and/or provides therapeutic treatment toan infected individual. As the VLP has adjuvant property, a vaccine ofthe invention has a tremendous immune-potentiating effect but with alower dose quantity than would be otherwise be necessary. Theconjugation reaction reduces the amount of antigenic material needed fora protective vaccination, preferably by about one half or less. Inaddition, the lack of a need for adjuvant, further reduces the amount ofmaterial necessary for a protective a vaccination. The reduction of bothantigenic materials needed and overall quantity required allows for theproduction of multivalent vaccines that are protective against largenumber of serotypes of a particular infectious agent, and also vaccinesthat are protective against multiple different infectious agents. One ofthe significant advantages attributed to the reduction of antigen neededand overall quantity of vaccine material is that vaccines of theinvention can be administered to children which generally require alower dose of antigen and/or vaccine quantity as compared with adultvaccines.

One embodiment of the invention is directed to VLP vaccines comprisingone or more and preferably multiple antigens of the same or multipledifferent infectious agents. Vaccines of the invention allow for addingall of the pathogenic serotypes, preferably of polysaccharides, making acomplete vaccine against the pathogen. The immunogenicity of such acombination is higher than that created from conventional multivalentvaccines, and with a reduced dose quantity which in turn would reducethe protein load to the patient. This is of particular importance wherethe conjugate vaccine includes CRM₁₉₇. The quantity of CRM₁₉₇ needed fora vaccine of the invention is quantitatively less by one half or more ascompared to conventional vaccines. Thus, VLP vaccines of the inventionavoid the major complications that prevent development of a completevaccine, and without creating either a toxic overdoes or any sideeffects attributed to the administration of high doses of an antigenand/or large volumes of a vaccine.

Virus-like particles resemble viruses, but are non-infectious and do notcontain viral genetic material. VLPs can self-assemble from expressionof the viral structural proteins, such as, for example, envelope orcapsid proteins in vitro or in culture. VLP portions are preferablycreated from virus or virus-like agents that infect humans, bacteria,parasites, fungus, plant, and/or other hosts. Preferred VLP that containviral proteins include, but are not limited to VLP's purified from orderived from hepatitis virus (e.g., Hep A, B, C, D and/or E), humanpapilloma virus (HPV), parvoviruses (e.g. 20 adeno-associated virus),respiratory syncytial virus, retroviruses (e.g. HIV), influenza virusand combinations thereof. VLPs of the invention are preferably obtainedor derived from Hepatitis B virus (Hep B) and composed of the small HBVderived surface antigen (HBsAg). VLPs can be produced in a variety ofcell culture systems including mammalian cell lines, insect cell lines,yeast, and plant cells. Preferably the VLP portion is a universallyrecognized portion of the infectious agent and may be purified,synthesized, created as fusion proteins, and/or recombinantly produced.Preferably the antigenic portions of the VLP are commercially availableand shown to be safe for use in vaccines having a recognized safetyprofile.

The immunogenic agent of the VLP comprises an antigen or other structurethat stimulates an immune response in an individual. Immunogenic agentsinclude, but are not limited to peptides, proteins, lipids, fatty acids,polysaccharides, lipopolysaccharides. Typically, immunogenic agents aresurface antigens of an infectious particle. Antigens may be specific fora particular infectious agent or combination of agents such as, forexample, viral infectious agents (e.g., enterovirus, hepatitis virus,human immunodeficiency virus {HIV}, human papilloma virus {HPV},influenza virus, pertussis virus, rubella virus, tetanus, varicellavirus {VZV}, flavivirus, West Nile virus, dengue virus, tick-borneencephalitis virus, yellow fever virus, Zika virus), bacterialinfectious agents (e.g., Chlamydia, Clostridium, diphtheria,meningococcal, streptococcal, staphylococcal, pneumococcal), parasiticinfectious agents (e.g., giardia, malaria {plasmodium}), and/or an agentthat causes sepsis or septicaemia. Preferably the immune response issufficient to protect the individual from subsequent infections for aperiod of time. Preferably the vaccine is effective and protects apatient from infection for six months or greater, one year or greater,two years or greater, five years of greater, or ten years or greater Theimmune response generated in response to the immunogenic agent of theinvention generates a humoral immune response, a cellular immuneresponse, or preferably both in the individual. Preferred cellularresponse includes a T cell response, and/or a phagocytic response, andalso preferably a memory cell response.

VLP of the invention may be fused, conjugated or otherwise attached(e.g., covalently or non-covalently bonded) with antigenic portions ofan infectious agent. Preferably, the VLP of the invention are conjugatedwith multiple polysaccharides (PS) of one or more infectious agents, andpreferably, capsular polysaccharides of the same or of differentinfectious agents such as, for example, infectious microorganism thatinfect humans. Conjugated vaccines may be created by covalentlyattaching a VLP to a PS using activating agents such as, for example,cyanylating reagents, such as, for example,1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP) or otherwell-known cyanylating reagent (e.g., 1-cyanobenzotriazole {1-CBT},2-cyanopyridazine-3(2H)-One {2-CPO}, 1-cyanoimidazole {1-CI},1-cyano-4-pyrrolidinopyridinium tetrafluoroborate {CPPT}). Processes forconjugation may include linker compounds and are very well know andcommercially available (Lees, et al., “Activation of SolublePolysaccharides with 1-Cyano-4-Dimethylamino PyridiniumTetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccinesand Immunological Reagents,” Vaccine, Vol. 14, No. 3 (1996), pp. 190198; U.S. Pat. No. 9,044,517). By coupling VLPs to multiple anddifferent PS (which may be of the multiple serotypes of the sameinfectious agent or multiple infectious agents), both strong and broadimmunity against an infection agent can be obtained as VLP's areinherently immunologic.

Preferably VLPs of the invention are coupled to all of the pathogenicforms (e.g., serotypes, strains, types, species, subspecies) of aparticular infectious microorganism, or at least all that are relevantto afford protection to an individual and/or particular population. PSmay be conjugated to the fully formed VLP or the structural componentsof the VLP and the VLP formed after conjugation.

In one embodiment, a conjugate vaccine can be created by covalentlyattaching an antigenic polysaccharide portion (which may have otherwisebeen found to be poorly antigenic) to a carrier protein, therebycreating the immunological attributes of the carrier also for theattached antigen. So the conjugated polysaccharide/protein carrierinduces a T cell response, but would be for both the constituents. Forexample, with cross-reactive antigen (e.g., CRM₁₉₇) as carrier, there isoften an enormous unwanted antibody generation towards the CRM₁₉₇. Suchis not the case when using VLPs as the immune response generated isprotective against the particular disease.

A VLP carrier protein conjugated with PS being highly immunogenicreduces the dose quantity otherwise required, while maintaining theimmune-potentiating power at reduced dose. Such a conjugate is stableand, optionally, can be formulated with aluminum hydroxide and phosphatebuffer for increased adsorption. Stability of VLP vaccines can befurther supplemented, without increasing the antigenic load, with theaddition of sugar alcohols such as, for example, sorbitol, gelatins andpreferably chemically and/or mechanically degraded gelatins (e.g., withaverage molecular weights of ten kilodaltons {kD} or less). Stabilizingagents that are useful with VLP vaccines are disclosed in U.S. patentapplication Ser. No. 15/257,143 entitled “VLP Stabilized VaccineCompositions” and filed Sep. 6, 2016, which is specifically incorporatedby reference.

A preferred multivalent conjugate vaccine of the invention comprises aplurality of capsular polysaccharides of Streptococcus pneumoniaeconjugated with a recombinant hepatitis VLP. The vaccine may beeffective against any or more, or all serotypes of Streptococcuspneumoniae (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, or 23 serotypes) Preferably the vaccinecomprises at least two different capsular polysaccharides representingat least two different serotypes of Streptococcus pneumoniae, morepreferably at least five, more preferably at least ten, more preferablyat least fifteen, more preferably at least twenty, and even morepreferably at least twenty three. The resulting vaccine is effective asa universal adult and also infant vaccine at least because the dosagerequired would be lower than currently available multivalentpneumococcal vaccine and would be well tolerated by children andinfants. The protein requirement as carrier would be around 23micrograms which is close to the adult dose. As no excess protein wouldbe injected, such vaccine would be much safer than is currentlyavailable. This allows for creation of a larger numbered polyvalentvaccine which otherwise due to large protein quantity may not beefficacious for all components.

Other infections agents include one or more of the eleven clusters ofStaphylococcus aureus and related sub-species, the twenty serotypes ofPseudomonas aeruginosa, at least three of which are clinically relevant(serotypes 03, 06 and 011), O antigen, K antigen and H antigen serotypesof Escherichia coli including pathogenic serotypes O157:H7, O104:H4,O121, O26, O103, O111, O145, and O104:H21, and the various pathogenicstrains and/or serotypes of Klebsiella, Enterobacter, Serratia,Citrobacter. Salmonella, Listeria, Shigella, Campylobacter, andMycobacterium.

Another embodiment of the invention is directed to the treatment and/orprevent of infection attributed to an infectious agent by theadministration of the VLP vaccine of the invention to a patient.Effective methods of administration of vaccines of the inventioninclude, but are not limited to administration via a patch (e.g., dermalpatch), injection (e.g., intra-muscular, intra-peritoneal, intra-organ,intra-venous, intra-cerebral), inoculation, oral or directgastrointestinal tract administration, transdermal absorption, a vaccinegun or jet injector or, preferably, through transdermal absorptionsusing dissolving or soluble biodegradable needle arrays. Preferably thevaccine of the invention is given as an intradermal injection or via adermal patch without aluminum adjuvants. Due to this route the dosequantity would be further reduced and yet having the vaccine aseffective and safe as the larger dose.

Preferably the vaccine is maintained as a liquid, but the liquid may belyophilized and stored as a dry powder until use wherein the powder isrehydrated in an appropriate carrying agent (aqueous or non-aqueous) foradministration to the individual. Administration is preferable byinjection which may be intra peritoneal (i.p.), intra muscular (i.m.),and/or intra venous (i.v.), or localized to the site of an infection.

Preferably the vaccines of the invention as liquids or powders arestable at 4° C. or greater, 15° C. or greater, 25° C. or greater, 37° C.or greater, 40° C. or greater, 50° C. or greater, or 100° C. or greater.Also preferably, the vaccines of the invention as liquids or powers arestable at 15° C. or less, more preferably to 0° C. or less, morepreferably to minus 20° C. or less, and more preferably to minus 50° C.or less. Also preferably, stability of the vaccines is maintained forsix month or greater, for eight months or greater or for twelve monthsor greater. Preferably the vaccines of the invention are stable throughvarying temperatures over time which may include multiple freezing andthawing.

Another embodiment of the invention is directed to methods for theadministration of vaccines of the invention to patients in need thereoffor treating or preventing an infection. The method comprisesadministering a therapeutically effective amount of the vaccine of theinvention to a mammal, comprising determining the therapeuticallyeffective amount of the vaccine to be administered that provides therapyto an infected patient and/or protection from infection. Thetherapeutically effective amount is typically determined by based on theweight of the mammal and the strength or responsiveness of the patient'simmune system and can be determined by those skilled in the art. Thetherapeutically effective amount is administered to a patient in needthereof, which may be to treat an active or suspected infection orprevent an infection. The vaccine may have been obtained from alyophilized powder and reconstituted to an aqueous or non-aqueous liquidprior to administration to the patient. Preferably the vaccine isadministered as a liquid, which may be intra-muscular, intra-peritoneal,or intra-venous, and the patient may be an infant, a toddler, anadolescent, an adult or a senior. Surprisingly, the vaccine of theinvention does not generate side effects such as redness or inflammationat the injection site, and does not generate a generalized fever orinflammation, or other unwanted side effects for the patient. Preferablyan immunologically effective vaccine contains only the fully formed VLPscontaining multiple immunogenic agents, and nothing further such as, forexample, no added adjuvants.

Another embodiment of the invention is directed to method for themanufacture of vaccines of the invention. Structural components ofviruses are obtained by methods well known to those skilled in the artand exclusive of any nucleic acid material that would allow thecomponents and resulting particles to replicate. Predetermined molaramounts of the structural components are mixed, preferably at roomtemperature or below, with approximately equivalent molar amounts of oneor more immunogenic agents and one or more stabilizing agents of theinvention, such that the VLPs encapsulate the one or more immunogenicagents and the one or more stabilizing agents in roughly equivalentamounts. The fully formed VLPs are separated from unformed VLPs and freestructural and other materials preferably by filtration, centrifugationor another method known to those skilled in the art, thereby creatingfully formed VLPs containing one more immunogens and one or morestabilizing agents. The fully formed VLPs may be stored as an aqueous(e.g., water or saline) or non-aqueous (e.g., oils, fatty acids)mixture, or lyophilized and stored as a powder. Preferably storage untiluse is without significant loss of immunogenic activity and, forexample, may be for one month or longer, four months or longer, sixmonths or longer, or more preferably one year or longer and at ambienttemperatures, whether as a liquid or a powder. Storage of vaccinewithout loss of immunogenic activity may also be at less than ambienttemperature such as, for example, at 20° C. or less, at 10° C. or less,at 4° C. or less, or at 0° C. or less.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

Example 1 VLP Conjugate Vaccine Against Streptococcus Pneumonia

VLPs are obtained from the structural components of hepatitis virus andcovalently conjugated with a capsular polysaccharide derived fromStreptococcus pneumoniae. The conjugated VLPs are coupled withStreptococcus pneumoniae immunogenic peptides, which are isolated fromthe microorganism or recombinantly prepared. The conjugate is formulatedwith aluminum hydroxide and phosphate buffer as an adjuvant and forincreased tissue adsorption, in accordance with the preparation ofconventional Hep B vaccines. The resulting conjugate vaccine is stablefor long periods of time (e.g., at least 1-6 months at 4° C. or at least12-24 months when lyophilized). Due at least in part to theimmune-potentiating power of the conjugate, the vaccine is administeredto a mammal at a dose of one microgram per serotype. With 23 serotypes,the protein load of the carrier is a total of about 23 micrograms pervaccination, which is close to the adult dose. When administered tomammal with an active immune system (e.g., an adult human), the patientgenerates a protective immune response to Streptococcus pneumoniae inthe form of both a humoral and a cellular response, and with no risk ofprotein overload.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.The term comprising, where ever used, is intended to include the termsconsisting and consisting essentially of. Furthermore, the termscomprising, including, and containing are not intended to be limiting.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

The invention claimed is:
 1. An immunogenic composition against multipleserotypes of an infectious agent comprising virus like particles andmultiple polysaccharides with little to no virus like particlemisassembly or polysaccharide misfolding, wherein the multiplepolysaccharides represent the multiple serotypes of the infectiousagent, wherein the infectious agent comprises Streptococcus.
 2. Theimmunogenic composition of claim 1, wherein the virus like particles areobtained or derived from hepatitis virus, human papilloma virus,respiratory syncytial virus, flavivirus or combinations thereof.
 3. Theimmunogenic composition of claim 1, wherein one or more of the multiplepolysaccharides are covalently coupled via conjugation to one or morecomponents of the virus like particles.
 4. The immunogenic compositionof claim 3, wherein the conjugation involves coupling via a cyanylatingagent.
 5. The immunogenic composition of claim 4, wherein thecyanylating agent comprises 1-cyano-4-(dimethylamino)-pyridiniumtetrafluoroborate (CDAP), 1-cyanobenzotriazole (1-CBT),2-cyanopyridazine-3(2H)-One (2-CPO), 1-cyanoimidazole (1-CI),1-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT) or a combinationthereof.
 6. The immunogenic composition of claim 1, wherein theinfectious agent comprises Streptococcus pneumonia.
 7. The immunogeniccomposition of claim 1, which does not require an adjuvant to generate atherapeutically effective response.
 8. The immunogenic composition ofclaim 1, further comprising an adjuvant.
 9. The immunogenic compositionof claim 8, wherein the adjuvant comprises aluminum hydroxide withphosphate buffer.
 10. The immunogenic composition of claim 1, which uponadministration to a patient, generates a therapeutically effectiveimmune response against the infectious agent.
 11. The immunogeniccomposition of claim 10, wherein the therapeutically effective immuneresponse comprises protection to an individual against a subsequentinfection of the infectious agent and/or therapeutic treatment to anindividual infected by the infectious agent.
 12. The immunogeniccomposition of claim 10, wherein the therapeutically effective immuneresponse provides protection against infection by multiple serotypes ofthe infectious agent.
 13. The immunogenic composition of claim 10,wherein the therapeutically effective immune response comprises ahumoral and/or a cellular immune response against the infectious agent.14. A vaccine against multiple serotypes of an infectious agentcomprising virus like particles coupled to multiple polysaccharidesand/or peptides with little to no virus like particle misassembly orpolysaccharide misfolding, wherein the multiple polysaccharide representthe multiple serotypes of the infectious agent, wherein the infectiousagent comprises Streptococcus.
 15. The vaccine of claim 14, wherein thevirus like particles are obtained or derived from hepatitis virus, humanpapilloma virus, respiratory syncytial virus, flavivirus or combinationsthereof.
 16. The vaccine of claim 14, wherein the multiplepolysaccharides and/or peptides are covalently coupled via conjugationto one or more components of the virus like particles.
 17. The vaccineof claim 16, wherein the conjugation involves coupling via a cyanylatingagent.
 18. The vaccine of claim 17, wherein the cyanylating agentcomprises 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP),1-cyanobenzotriazole (1-CBT), 2-cyanopyridazine-3 (2H)-One (2-CPO),1-cyanoimidazole (1-CI), 1-cyano-4-pyrrolidinopyridiniumtetrafluorborate (CPPT) or a combination thereof.
 19. The vaccine ofclaim 14, wherein the infectious agent comprises Streptococcuspneumonia.
 20. The vaccine of claim 19, wherein the virus comprises oneor more of enterovirus, hepatitis virus, human immunodeficiency virus(HIV), human papilloma virus (HPV), influenza virus, pertussis virus,rubella virus, tetanus virus, varicella zoster virus (VZV), flavivirus,West Nile virus, dengue virus, tick-borne encephalitis virus, yellowfever virus, Zika virus and combinations thereof.
 21. The vaccine ofclaim 14, which does not require an adjuvant to generate atherapeutically effective response.
 22. The vaccine of claim 14, furthercomprising an adjuvant.
 23. The vaccine of claim 22, wherein theadjuvant comprises aluminum hydroxide with phosphate buffer.
 24. Thevaccine of claim 14, which upon administration to a patient, generates atherapeutically effective immune response against the infectious agent.25. The vaccine of claim 24, wherein the therapeutically effectiveimmune response comprises protection to an individual against asubsequent infection of the infectious agent and/or therapeutictreatment to an individual infected by the infectious agent.
 26. Thevaccine of claim 24, wherein the therapeutically effective immuneresponse provides protection against infection by multiple serotypes ofthe infectious agent.
 27. The vaccine of claim 24, wherein thetherapeutically effective immune response comprises a humoral and/or acellular immune response against the infectious agent.
 28. Theimmunogenic composition of claim 1, wherein the multiple polysaccharidesare obtained and/or derived from five or more of the serotypes 1, 2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F, and 33F of Streptococcus pneumoniae.
 29. The immunogeniccomposition of claim 1, wherein the multiple polysaccharides areobtained and/or derived from ten or more of the serotypes 1, 2, 3, 4, 5,6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F,23F, and 33F of Streptococcus pneumoniae.
 30. The immunogeniccomposition of claim 1, wherein the multiple polysaccharides areobtained and/or derived from fifteen or more of the serotypes 1, 2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F, and 33F of Streptococcus pneumoniae.
 31. The immunogeniccomposition of claim 1, wherein the multiple polysaccharides areobtained and/or derived from twenty or more of the serotypes 1, 2, 3, 4,5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F, and 33F of Streptococcus pneumoniae.
 32. The immunogeniccomposition of claim 1, wherein the multiple polysaccharides areobtained and/or derived from twenty three or more of the serotypes 1, 2,3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,20, 22F, 23F, and 33F of Streptococcus pneumoniae.